yeast homologs of the mammalian checkpoint …pressing element, called a tsl (for alpha tubulin...

Copyright 0 1994 by the Genetics Society of America

Overexpression of Yeast Homologs of the Mammalian Checkpoint Gene RCCl Suppresses the Class of a-Tubulin Mutations That Arrest With

Excess Microtubules

David Kirkpatrick' and Frank Solomon

Department of Biology and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 0 2 1 3 9

Manuscript received December 8, 1993 Accepted for publication February 23, 1994

ABSTRACT Microtubules in eukaryotic cells participate in a variety of nuclear and cytoplasmic structures, reflecting

functional requirements and cell cycle position. We are studying the cellular regulation of microtubule assembly and organization in the yeast Saccharomyces cerevisiae. We screened for genes that when overexpressed suppress the growth phenotype of conditional mutants in a-tubulin that arrest with excess microtubules at the nonpermissive temperature (class 2 mutations). Here we describe one such sup pressing element, called A T S l (for Alpha Tubulin Suppressor). Overexpression of this gene rescues both the growth and microtubule phenotypes of all class 2 mutations, but not the cold-sensitive mutations that arrest with no microtubules (class 1 mutations). Deletion of ATSl confers a modest slow growth phenotype which is slightly enhanced in strains containing both a deletion of ATSl and a class 2 tub1 mutation. The predicted A T S l protein contains 333 amino acids and has considerable structural homology to the prod- ucts of both the mammalian mitotic control gene RCCl and the S. cerevisiae gene SRMl/PRPBO. Over- expression of SRMl/PRPBO also suppresses class 2 mutants. The results suggest that this family of genes may participate in regulatory interactions between microtubules and the cell cycle.

M ICROTUBULES in the budding yeast Saccharomy- ces cerevisiae are found in both the cytoplasm and

the nucleus, and are essential for chromosome segre- gation (MEEKS-WAGNER et al. 1986) and nuclear move- ments (HUFFAKER et al. 1988). There are several possible mechanisms for cellular specification of the repertoire of microtubule functions and structures. In yeast, nu- merous experiments argue that neither the primary structures of the tubulin proteins nor quantitative regulation of tubulin protein levels are major regulatory factors (SCHATZ et al. 1986; KATZ et al. 1990). A third possibility is that associated proteins and structures may control the timing, organization and interactions of microtubule assembly. Genetic methods for identifjmg such proteins, screens for either pseudo-reversion of conditional mutations in the tubulin genes (SCHATZ et al. 1988) or unlinked noncomplementation of such mutations ( STEARNS and BOTSTEIN 1988), yield only mutations in the tubulin genes themselves. Approaches that focus directly on processes known to involve microtubules have been more successful in identdjmg putative microtubule associated proteins such as microtubule based motors (see, for example, MELUH and ROSE 1990; SAUNDERS and How 1992) (reviewed in EPSTEIN and SCHOLEY 1992), and can- didates for components of the yeast microtubule organiz- ing centers, the spindle pole bodies (SPBs) (see, for example, PAGE and SNYDER 1992; WINEY et al. 1991).

Chapel Hill, North Carolina 27599-3280. I Present address: Department of Biology, University of North Carolina,

Genetics 137: 381-392 (June, 1994)

We have taken an alternative approach to this prob- lem, based on observations suggesting that factors other than tubulin concentration are limiting for microtubule assembly. To identify such factors, we screened for genes that, when overexpressed, rescue conditional mutations in a-tubulin that cause arrest with excess microtubules. This phenotype (class 2 mutants; SCHATZ et al. 1988) occurred in a substantial fraction of cold-sensitive mutants of a-tubulin generated by chemical mutagenesis protocols. The same mutagenesis also produced mutants that arrest with no microtubules (class 1 mutants), or with no conspicuous quantitative defect (class 3 mutants). Here we show that overexpression of one gene, called A T S l , rescues five independent class 2 a-tubulin mutations. Excess A TSl restores nearly normal growth and microtubule morphology to these mutants at the restrictive temperature. However, excess ATSl has no effect on class 1 a-tubulin mutants that arrest at the same temperature. We demonstrate that ATSl is structurally and functionally related to a family of putative mitotic checkpoint genes that include the mammalian RCCl. The data suggest a role for A TSl in the coordination of microtubule state with cell cycle regulation.

MATERIALS AND METHODS

Strains and media: Yeast strains used in this study are indicated in Table 1. Media, both rich (YPD) and synthetic complete (SC) and minimal (SD) , were prepared as described in SHERMAN et al . (1986). Unless indicated otherwise, all strains are derived from DBY strains, in the S288c background.

382 D. Kirkpatrick and F. Solomon

TABLE 1

Strains and plasmids

Strain Genotype Source

DBE403

DBY2412 DBY2414 DBY2418 DBY2426 DBY2433 FSYl20 FSYl85

FSY340 FSY34 1 FSY342 FSY343 FSY336 FSY332 FSY337 FSY338 FSY344 FSY345 FSY346 FSY347 FSY348 FSY349 FSY350 FSY35 1 FSY352

ASY257 FSY353

NMYl F63-7d

MATa his?-A200 leu2-?,112 lys2-801 urn?-52 tubl::HIS? tub3::TRPl

As DBY2403 except tubl-730 (pRB630) As DBY2403 except tubl-733 (pRB633) As DBY2403 except tubl-741 (pRB641) As DBE403 except tubl-758 (pRB658) As DBY2403 except TUBI (pRB658) MATa/a his4-619/+ leu2-3,112/leu2-3,112 lys2-801/+ ura2-52/uru3-52 MATa/u ade2/+ his3-A200/his?-A200 leu2-?,112/leu2-3,112 lys2-801/lys2-801

urn3-52/ura3-52 DBY2414 with pDK7 DBY2414 with pDKI6 DBY2414 with pDK32 DBY2412 with YCp50 DBY2412 with pDK7 DBY2412 with pDK8 DBY2412 with PDK32 DBY2412 with pWF48 FSYl85 with pLGSD5 FSYl85 with pDK33 DBY2403 with pDK44 and pRB614 DBY2426 with pDK44 and pRB658 FSY346 but Aats1::URAjr FSY346 but URA3 FSY349 with only pDK44 (wild type for TUBl and A T S l ) FSY348 with only pDK44 (wild type for TUBI, Aats l ) FSY349 with only pRB614 (tubl-714, wild type for A T S l ) FSY348 with only pRB614 ( tubl-714, Aats l ) tub2-412:URA? ura?-52 ude2 his? lys2 tub2-150 urn?-52

tubl-714-LEU2-CEN4-ARSl (pRB614) SCHATL et al. (1988)

SCHATZ et nl. (1988) SCHATZ et nl. (1988) SCHATZ et nl. (1 988) SCHATZ et al. (1 988) SCHATZ et nl. (1988) KATZ and SOLOMON (1988) KATZ et al. (1990)

This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study T. HUFFAKER G. BARNES

MATa prpZ0-2 ura?-52 W. FORRESTER

Plasmid

YCp50 pDK7 pDK8 pDK9 pDKlO pDKl6 pDK31 pDK32 pDK33 pDK39 pDK44 pLGSD5 pWF48

Description

CEN4 ARSl URA3 CEN4 ARSI URA3 TUBl CEN4 ARSl URA3 suppressor 1 CEN4 ARSl URA3 suppressor 2 CEN4 ARSl URA3 suppressor 3 CEN4 ARSl URA3 ATSl pBR322 derivative: Aatsl::URA3 2 y m URA3 ATSl pLGSD5 derivative: 2 y m URA3 GALl, 10-CYCI-ATS1 CEN4 ARSl HIS3 ATSl CEN4 ARSI LYS2 TUBl 2-pm URA? GAL1,lO-CYCl-LACZ 2-urn URA? PRP20

Source

This study This study This study This study This study This study This study This study This study This study GUARENTE et al. (1982) W. FORRESTER

Standard manipulations (SAMBROOK et al. 1989, SHERMAN et al. 1986) and those described in SOLOMON et al. (1992) were used.

Isolation of suppressors: Transformations were performed using the LiAc method, as detailed in SOLOMON et al. (1992), modified from ITO et al. (1983). Transformations to evaluate suppression of cold-sensitive mutations were plated on SC dropout plates and placed at room temperature and at 15" unless indicated otherwise. For other transformations, cells were grown at 30" after plating.

Plasmid loss assay: Strains were grown in 5 ml liquid WD media for 24 hr after inoculating from colonies on plates. A 100-pl aliquot of the overnight culture was added to 5 ml WD and grown for 24 hr again. Cell number was determined by hemocytometer counts, and appropriate dilutions were plated to WD or SC plates and incubated at 30" until colonies were visible. The colonies were then replica-plated to SC-uracil, SC- leucine or SC-lysine, depending on the strain. A loss event, indicated by the absence of the particular plasmid, was scored

if a colony was totally absent on the drop-out plate; partial colonies were not counted.

Sequencing: Sequencing of both strands of the region containing ATSl was performed utilizing modified T7 DNA polymerase Sequenase with the dideoxy chain termination method (U.S. Biochemical Corp.). A combination of sub- clones in the pSK+ and pKS+ Bluescript vectors (Stratagene) and oligonucleotides directed to various internal sequences of A TSl was utilized to generate multiply overlapping sequences from both strands of the ATSl gene. Oligos used were: top 2, CGCGTGTTGGCGGATGT (961-978); top 3, CGGGCCAA- CAACGAAAA (1 132-1 148) ; top 4, CTGGAGGCCCGTGGA- AG (237-253); top 6, ACTGAITTAAGTTACGT (-187 to -171); bottom 1 CTTATCAGTGCTAGAGC (1013-997); bottom 2, ACCGTGCTCACTCCCGG (820-804); bottom 3, GCT- GTTGT'ITGAGCTCG (652-636); bottom 4, GTCCAGCCG CACGCCAC (291-274). Location is given relative to the ATG in the ATSl open reading frame, with A = 1. "Top" oligos

High Copy Tubulin Suppression 383

hybridize to the nonsense strand, "bottom" to the sense strand. Two oligos have minor inconsistencies with the actual genomic sequence: top 2 is missing a T at the 7th position compared to the actual ATSl sequence; bottom 4 is missing a C at the 3rd position.

Growth rate determinations: Growth rates were determined by inoculating 5-ml cultures of the appropriate SC media with a fresh colony or a frozen stock and incubating at 30" or room temperature overnight on a rotating drum. Cell density was determined by counting with a hemocytometer. Samples of 10 ml of defined media in 125-ml flasks were in- oculated with 1 X lo5 to 5 X lo5 cells/ml, and placed at the desired temperature. Cell number was evaluated by hemocytometer counts, while viable cell number was determined by plating dilutions (based on hemocytometer counts) on selective media and incubating at 30".

Immunofluorescence: Immunofluorescence studies of the microtubule morphologyand DNAcontent of strainswere performed as described previously in SOLOMON et aZ. (1992). Pri- mary antibodies used were anti-Ptubulin rabbit polyclonal an- tisera, 2061 and a monoclonal antibody against a-tubulin, AlBG7. Secondary antibody was fluorescein isothiocyanate- conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Cap pel). 4',6Diamidino-2-phenylindole dihydrochloride (DAPI) (Boehringer Mannheim) was used to visualize DNA. A Zeiss Axioplan microscope was utilized for all immunofluorescence.

Plasmid constructions: Standard molecular biological methods (SAMBROOK et al. 1989) were used in generating re- combinant plasmids. Restriction enzymes and ligases were ob tained from New England Biolabs, Inc., and supplied instruc- tions were followed unless stated otherwise. Plasmids used in this study are listed in Table 1.

Bud size distributions: Bud sizes were determined in the following manner. An overnight culture of the strain was grown at 30" in SC dropout media. Cell densitywas determined by hemocytometer count and appropriate dilutions made. Cells were placed at the desired temperature and allowed to grow until mid-log phase. Cell density was counted and bud size scored.

Overexpression construction: pLGSD5 (GUARENTE et al. 1982) was digested with BamHI. A BclI to BglII DNA fragment of 1.1 kb containing the ATSI-coding region was ligated into this BamHI site such that 24 bp separate the ATG in ATSl from the CYCl leader of pLGSD5. Insertion of the ATSl fragment was possible in either orientation-the direction of insertion was determined by diagnostic restriction digests with EcoRI and HzndIII. pDK33 contains ATSl in the correct orientation; pDK34 in the reverse orientation.

Deletion construction: YEp24 was digested with EcoRI and ligated, removing the 2ym sequences but retaining the URA3 gene. This derivative was digested with BamHI and SphI and ligated to a 300-bp BglII-SphI fragment containing noncoding sequences 3' of the ATSI gene. Finally, this construct was digested with CZaI and SspI, and ligated to a CZaI to HincII fragment containing 5"noncoding ATSI sequence. This generated a plasmid containing a URA3 gene flanked by 5'- and 3"noncoding ATSl sequence. Digestion with XmnI and SphI yields a linear fragment of approximately 1880 bp with ends derived from ATSl locus sequence.

RESULTS

Phenotypes associated with the class 2 tubl mutants: SCHATZ et al. (1988) used a plasmid shuffle to introduce mutagenized TUBl sequences into a haploid yeast strain (DBY2384) in which the chromosomal copies of both a-tubulin genes, TUBl and TUB3, were deleted. Among

those strains unable to grow at 11' were 5 independent isolates that displayed extra microtubules 24 hr after shift to the restrictive temperature, as assayed by immunofluorescence. Most conspicuous are the cytoplasmic microtubules, which traverse the full length of the cell or make complete circuits around the cell perimeter. The spindle microtubules also appear to be more in- tensely stained. The extra microtubules are not the result of increases in the level of tubulin protein. Neither a- nor Ptubulin levels, measured by western blotting, are elevated in the extra-microtubule mutants relative to wild-type strains at the nonpermissive temperature (L. CONNELL, unpublished observations). Also at 24 hr after shift to nonpermissive temperature, between 52 and 82% (depending on the allele) of the cells arrest with a large bud. These phenotypes largely persist at 15", the highest restrictive temperature for these mutants. In DBY2412 ( tubl - 730) approximately 60% of the cells arrest with a large-budded morphology at 15" (see Figure 3B for example).

Suppression of extra-microtubule mutants by overexpression: DBY2412 (tubl-730) was transformedwith a yeast genomic DNA library (ROSE et al. 1987) borne on a YCp50 plasmid bearing the selectable marker URA3. Three of 3200 transformants grew at 15" after successive platings. We isolated the URA3 genomic library plasmids from each of those strains (pDK8, 9, 10). Re- transformation of DBY2412 with each of the plasmids confers cold resistance. In addition, when such transformants are allowed to lose the URA3 plasmid by growth in medium containing uracil at the permissive temperature (see MATERIALS AND METHODS), they no longer can grow at 15". Therefore, the plasmids themselves are responsible for suppression of the cold- sensitivity associated with tubl- 730. Transformants incubated at 15" indicated that the three plasmids were not identical in their ability to suppress the cold sensitivity of DBY2412. pDK8 demonstrated the greatest suppressing activity based on colony size and number of transformants obtained at the nonpermissive temperature (see Figure 1 ) .

Cloning and sequencing of suppressor: A restriction map of the 6-kb genomic insert of pDK8 was generated and used in subcloning the region. The suppressing element was localized to a 1.3-kb ClaI to SphI fragment. This region contains an open reading frame of 999 nucleotides which we have designated A TSl (Alpha Tu- bulin Suppressor). Computer analysis of the putative Atslp predicts no long stretches of a helices or p sheets, nor does it seem to possess a membrane-spanning domain. Microtubule-binding sites from mammalian cells (NOBLE et al. 1989; LEE et al. 1988; and LEWIS et al. 1988) are also not present. The ATSl protein possesses seven internal repeats that constitute the entire open reading frame (Figure 2).

384 D. Kirkpatrick

2 2 O 15'

FICURF. 1 .--Suppression of t h r cold scnsitivitv of DRY2412. The uppermost block of photographs shows selective plates containing DRY2412 ( t u b l - 7 3 0 ) transformed with the vector YCp50 and incubated at either 15" or room temperature for 2 weeks. The second block shows DBE412 transformed with pDK8 ( A T S I ) . pDK8 showed the greatest extent of suppression, and was further characterized.

Characterization of ATSl suppression: Allele speci- ficity: ATSI was identified by its ability to suppress the cold sensitivity of t u b l - 7 3 0 , which undergoes growth arrest at 15". The A TSI sequence also allows the other four class 2 mutants to grow at the restrictive temperature. It is important to note that the 5 class 2 mutants were generated independently of one another. They map to three different domains of the TUBl gene; those that are in the same domain derive from separate mutagenesis reactions (SCHATZ et a l . 1988). Further experiments indicate that A TS1 does not suppress the cold sensitivity of class 1 mutants, which exhibit no microtubules at the nonpermissive temperature (see Table 2). We conclude that suppression by excess ATSI is not general for t u b l mutations, but rather specifically suppresses the cold- sensitive phenotypes of the mutant t u b l alleles that dis- play an excess of microtubules upon shift to the restrictive temperature.

Microtubule morphology: The extent of suppression of aberrant microtubule structures was examined by indirect immunofluorescence after 24 hr at 13" (Figure 3A) in DBM414 (this strain was used rather than DBM412 due to the high background staining of DBY2412 with our antibodies; excess ATSI suppresses the cold sensitivity of DBY2414). Approximately 50% of cells bearing the t u b l - 7 3 3 mutation had aberrantly large microtubule structures. These excess microtubules

and F. Solomon

were greatly reduced upon transformation with pDK7 (CEN TUBI) . Extra-microtubule tubl mutant strains containing pDK7 grow at the same rate at 15" as strains wild type at the TUBl and TUB3 loci. Increased levels of ATSI, borne on either low copy ( CEN) or high copy (2ym) plasmids, suppress the microtubule defects in DBY2414. The extent of ATSI suppression is comparable with both CEN and 2ym plasmids. That the microtubules of the suppressed mutant cells are not fully identical to mutant cells containing pDK7 ( CEN TURI) suggests that above a certain level of ATSI protein, another factor becomes limiting for suppression.

Bud size distribution: Analysis of DBM412 cells containing a plasmid bearing no insert, the TUBl gene, or ATSl demonstrated that extra copies of ATSI are capable of restoring a normal pattern of bud size distribution to DBY2412 cells at the nonpermissive temperature (Figure 3B). Thus, by two criteria in addition to restoration of growth at 15", additional copies of ATSl are capable of suppressing defects associated with the tubl extra- microtubule mutants.

Does excess ATSl induce microtubule loss in wild- type cells?: If the suppression of the extra-microtubule mutants is due to ATSI acting as a destabilizer of microtubules, then overexpression of ATSI in wild-type cells might prove deleterious through reduction of normal microtubule structures. To test this model, we determined the growth rate for wild-type strains bearing extra A TSI. Excess A TS1 was produced either through introduction of low or high copy plasmids, or through galactose-regulated overexpression. The rate of growth for wild-type DBY2433 cells did not vary when extra ATSl was present (Figure 4). Microtubules in these strains appeared normal by indirect immunofluorescence. To look for more subtle alterations, we examined bud size distribution to determine if the presence of excess A TSI protein altered transit through stages of the cell cycle. Transformants containing pDK32 (2ym ATSl ) and assayed at 22" showed no significant differ- ences in bud size distributions as compared to control strains (Figure 4B). All of these experiments indicate that excess A TSI has no specific effect on wild-type cells.

In class 1 mutants, overexpression of a protein that down-regulates microtubule number or extent might ex- acerbate the nemicrotubule phenotype, leading to a synthetic lethality at formerly permissive temperatures. To determine if overexpression of ATSl was actually deleterious to class 1 mutants, 1 1 class 1 mutants were transformed with the A751 galactose-inducible construct pDJS33. No alter- ation in the severity of the cold sensitivity associated with any class 1 mutants of TUBl was observed when A751 was overexpressed in those cells (see Table 2).

Effect of excess ATSl on tub2 mutants exhibiting excess microtubules: Can ATSI suppress other mutants with phenotypes reminiscent of those of the class 2 extra- microtubule mutants? Two strains, "57 ( t u b 2 - 4 1 2 )


FIGURE 2.-ATSI protein residues arranged as a series of seven tandem repeats. Dashes indicate spaces inserted to maximize the similarities between repeats. Each repeat is approximately 50 amino acids long. Boxed amino acids are those conserved between repean. Repeats were aligned by utilizing the Genetics Computer Group program "Compare" and by visual inspection of the alignment of RCCl protein repeats (OHTSLIRO nl. 1987).

TABLE 2

Excess ATSl suppresses class 2 tub1 mutations

Suppression with

Class 2 alleles 15" 30" 15" 30"

Vector T U B l A TS1

lubl-714 - +++ +++ +++ ++ +++ luhl-730 - +++ +++ +++ ++ +++ IUhI-733 - +++ +++ +++ ++ +++ lllbl-741 - +++ +++ +++ ++ +++ tubl-758 - +++ +++ +++ ++ +++

Suppression with

Vector TUB 1 A TSI

Class 1 alleles 15" 30" 15" 30" 15" 30" 15" 30" 15" 30" 15" 30"

A B A B A B

++ +++ ++ +++ +++ +++ +++ +++ +++ ++ +++ +++ +++ +++ +++

ND ND ND ND ND ND ND ND ND ND ND

ND ND ND - ND - ND ND ND ND ND ND ND

++ +++ ++ +++ +++ ++ +++ +++ +++ +++ +++

Excess ATSI suppresses class 2 tubl mutations. Strains bearing class 2 extra microtubule alleles were transformed with a vector alone (YCp50). pDK7 (CEN T U B l ) or pDK8 (CEN A T S I ) and incubated at 15" or 30" to assay extent of suppression. Two experiments were conducted with strains bearing class 1 nwnicrotubule alleles to assess the ability of A T S l to suppress the cold sensitivity of class 1 mutants. In column A, the indicated strains were transformed in the same manner and with the same plasmids as for the class 2 strains. In column B, strains were transformed with pLGSD5 [vector with rnlactosidase under the control ofthe G A L I , loupstream region (GUARENE el al. 1982)] or pDKX? ( G A L ) , I O A T S I ) and extent of suppression was determined on galactosezontaining plates at 15" or 30" (ND = not done: galactose-mediated overexpression of TUB1 is detrimental (WEINSTEIN and SOLOMON 1990) and so w a s not used in this experiment.)

and NMYl (tub2-150), were obtained from TIM HUFFAKER and GEOR~IANA BARNES. NMYl is a @tubulin mutant that requires benomyl and generates elongated cytoplasmic microtubules (T. HUFFAKER, personal communication). Overexpression of A TSI in this strain did not alter the benomyl phenotypes or the temperature sensitivity associated with this strain. Likewise, the cold sensitivity associated with ASY257 did not change when ATSl was introduced, although this mutant strain also generates excess cytoplasmic microtubules at the nonpermissive temperature (G. BARNES, personal communication). Therefore, ATSl is not a general suppressor of mutants that exhibit enhanced microtubule arrays.

Genetic interactions between ATSl and TUBl: Com- plete elimination of the coding region of ATSl in h a p loid cells still permits growth under all conditions tested. However, we observe a reduction in growth rate at high and low temperature in strains with a deletion of ATSI, as indicated by tetrad analysis of spores from diploid strains heterozygous for the ATSl deletion, after ger- mination and growth at 11 O or 37" (Figure 5A). To look for possible synthetic interactions between ATSI and TUBl we attempted to delete the chromosomal copy of ATSI in the extra-microtubule tubl mutant background using a Aatsl::URA3 construct. Initially a TUB1 LYS2 CEN plasmid (pDK44) was introduced into DBY2403 ( tub l -714) , to complement the tubl muta-

D. Kirkpatrick and F. Solomon

20-

10 -

T- A T T

DBY2414 DBY2414 + pDK7 DBY2414 + pDK16 DBY2414 + pDK32

El Small Bud El LargeBud

3+ Cells

FIGURE S.-Excess A TSI suppresses phenotypes associated with Class 2 mutations. (A) Microtubule morphology of DBY2414 ( t u b l - 7 3 3 ) and suppressed strains. Cells were examined for microtubule morphology after 20 hr at 13". The following classes were scored: cells with no microtubules, cells with a single dot of staining, cells with a short bar ( a p proximately one-half the cell diameter) or in budded cells with a spindle, and finally cells with large structures (greater than one-half the cell diameter) or budded cells containing a spindle and with large cytoplasmic microtubules. pDK7 is a QTV TUUl plasmid, pDKl6 is a CEN ATSI plasmid, and pDK32 is a 2-pm AT.72 plasmid. (B) Distribution of bud sizes in DBM412 (tubl-730) transformed with various plasmids and amyed at 15" after 20 hr. DBM412 was transformed with YCp50, pDK7 (TUB]) , pDK32 (2-pm AT.71) or pWF48 (2-pm PRP20). Buds were scored as either no bud, small (less than 75% of the mother), large (greater than 75% of the mother) or three or more cells together.

DBY2412 + YCpSO DBY2412 + pDK7 DBY2412 + pDK32 DBY2412 + pWF48

tion. The resulting strains contained CENplasmids bearing both mutant and wild-type versions of TUBI.

Southern blotting identified successful deletions of ATSI. Plasmid loss (see MATERIALS AND METHODS) was then used to isolate strains containing either pDK44 (TUBI) or pRB614 ( tubl-714) , in both the ATSl and Aatsl backgrounds. Growth rates at 30" were determined for these four strains. FSY353, bearing both the deletion of ATSl and the tubl-714 allele, doubles 10- 20% slower than the other strains; 109 minutes for the double mutant as compared to 89 min for an ATSl

TUBI strain. Single mutants in TUB1 or ATSl double approximately every 97 min. Also, those colonies generated by the double mutant are smaller than the wild- type strain (Figure 5B). Our experiments indicate a mild reduction in growth rates for cells doubly mutant for a deletion of ATSl and tubl-714, but no drastic phenotype associated with the double mutant.

Structural homologs of ATS1: Computer analysis of the ATSl open reading frame indicated that the 333 amino acids were organized as a set of seven tandem repeats of approximately 50 amino acids each (Figure


A --C DBV2433 + pDK32 #2 * DBV2433 + pDK32 X1

lo6:

10'

107

l o 6

10'

104 0 2 4 6 0 1 0 1 2 0 2 4 6 0 1 0 1 2

Time (hours)

"1

60] B 0 Small Bud

Ll Large Bud 0 3+ Cells

DBY2433 + VCp50 DBY2433 + pDK7

2). Alignment of these repeats allowed identification of residues conserved between repeats. Probing of protein databases with the ATSl polypeptide sequence revealed similarities between Atslp and a set of homologous proteins from diverse organisms. The best match was to the mammalian protein, RCCl (OHTSUBO et al. 1987) (Fig- ure 6A). RCCl also contains seven tandem repeats, with the amino terminal repeats the most conserved. An- other homolog to RCCI has been identified in S. cerevisiae, called either SRMl (Cwuc and SPRAGUE 1989) or PRP20 (AEBI et al. 1990; FLEISCHMANN et al. 1991). It is an essential gene encoding a protein of 482 amino acids, arranged as seven tandem repeats.

Comparison of pairwise combinations of the Atslp repeats with one another utilizing the Genetics Com-

1"

DBV2433 + pDK32

FIGURE 4.-Wild-type strains are not af- fected by excess ATSl. (A) Excess ATSl does not affect doubling times of wild-type strains at 22". A strain isogenic with the extra-microtubule mutant strains but wild-type at TUBl (DBY2433) was transformed with pDK32 (2ym A T S l ) or YCp50. Two independent isolates of each were grown in liquid medium at 22". Strains containing excess ATSl did not significantly differ in growth rate from vectorcontaining strains. (B) Excess ATSl does not alter bud size distributions in wild-type cells. DBY2433 was transformed with YCp50, pDK7 ( T U B l ) or pDK32 (2-pm A T S l ) . Cells were grown, then diluted and incubated at 22" for 21 hr. Buds were scored as either no bud, small (less than 75% of the mother), large (greater than 75% of the mother) or three or more cells together.

puter Group program "Bestfit" indicates that the similarity between repeats varies from 27 to 67%, with an overall similarity of 50%. Comparisons for Prp2Op indicate a range from 39 to 62% (overall equal to 52%). Pairwise combinations of Atslp and Prp20p range from 29 to 63%. In summary, ATSl protein repeats are roughly as similar to themselves as they are to PRP20 protein repeats, and vice versa (Table 3).

Sequence comparison localizes ATSl to a recently se- quenced l&kb tract of chromosome I of S. cerevisiae (CLARK et al. 1992; OUELLETTE et al. 1993 GenBank ac- cession no. L05146). In that study ATSl is referred to as a "Function Unknown Now" gene, FUN28, and the similarity to RCCl and its homologs is noted. No lethality associated with an amino terminus deletion of FUN28


was detected; conditional phenotypes were not assayed. The ATSI nucleotide sequence differs from the pub- lished sequence at two nucleotides: CG becomes GC, changing an alanine (amino acid 305) to a glycine.

Functional relationships between ATSl and PRP20: To test the possibility that A T S l and PRP20 are functionally related, we transformed DBY2412 with PRP20.

A

t

B ATSI TUB1 ATSI t~bl-714

Aatsl::URA3 TUB1

The transformants grew at 15" (Figure 6B). Analysis of bud size distributions for these transformants demonstrated that extra copies of PRP20 are capable of restoring a normal pattern at the nonpermissive temperature, in a manner similar to A T S l (see Figure 3B). By that criterion (suppression of the class 2 tubl mutants), PRP20 and A T S l possess similar functions. Conversely, the temperature sensitivity of a strain bearing the prp20-2 mutation was unaffected by galactose-regulated overexpression of A T S l at either 33" or 37". A further test for interaction was performed by crossing haploids containing the Aatsl::URA3 allele with a prp20-2 ura3-52 haploid strain followed by tetrad dissection to obtain haploid prp20-2 Aatsl mutants. The prp20-2 Aatsl::URA3 double mutants were viable and displayed a temperature-sensitivity comparable to that of the prp20-2 mutants alone. These results indicate that although both A T S l and PRP20 are capable of suppressing the phenotypes associated with the class 2 mutants they are not functionally interchangeable in vivo.

DISCUSSION

In this study we report the identification and characterization of a quantitative suppressor of the a-tubulin class 2 extra-microtubule mutants. The distinguishing property of these conditional mutants is that they arrest with excess microtubules. The cold sensitivity, large budded arrest and excess microtubule phenotypes are greatly reduced in strains containing additional copies of the suppressor A T S l , which codes for a protein of 333 amino acids. Our results demonstrate that A T S l is not an essential gene. However, it has structural and functional homology to a gene that is essential, S R M l / PRP20. Deletion of A T S l in class 2 mutants results in a moderate reduction in the growth rate.

Quantitative suppression analyses are based on the hypothesis that the excess product will act within its normal domain of function, but that the excess will facilitate that function, thereby compensating for the defect in

FIGURE 5.-Deletion of ATSI confers modest growth phenotypes. (A) Colonies resulting from tetrads dissected and incubated at extreme temperatures. A diploid strain heterozygous for Aats::URA3 was sporulated and asci dissected and incubated at 11" (top) or 37" (bottom). At 1 lo , 43 spores formed colonies. Of these, 14 are noticeably smaller than their siblings. All 14 are Ura+. For example, tetrad 1 (from the left) contains two Ura+ (la, b) and two Ura- (IC, d) colonies. Tet- rad 3 (from the left) is an aberrant tetrad: 3a is Ura-, 3b-d are Ura+. At 37" of 31 spores, 17 were smaller, and of these, 14 were Ura'. As an example, tetrad 8 (rightmost) contains two Ura' (8b, d) and two Ura- (8a, c) colonies. (B) An atsl tubl mutant strain generates small colonies. Cells were grown in liquid media and then dilutions plated on selective plates and incubated at 30" for 2 days. FSY350 ( A TSl TUB1 ) is shown in the upper right, with FSY351 ( A n t s ] TUB1)beneath; FSY352 (ATS1 tubl-714) is shown in the upper left and FSY353 (Ants1 tubl- 71 4 ) beneath. FSY353 colonies are noticeably smaller in comparison to the other three strains.


A - T L G P G D V - G P L G L G E N V M - E R K K - P - - - - A L V S I P E D V V - - - - - - - - - - - - - - - - - - P A E A G G M H T V C L S K S G Q V Y

S F R D N N G V I G L L E P - M K K S - - - M V - P - - - - V P V O L D V P V V - - - - - - - - - - - - - - - - - - K V A S G N D H L V M L T A D G D L Y - S F G C N D E - G A L G R D T S V E G S E M V - P - - - - G K V E L G E K V V - - - - - - - - - - - - - - - - - - P V S A G D S H T A A L T D D G R V F L W G

- T L G C G - E P G Q L G R V P E L F A N R G - - - - - - - G R O G L E R L L V P K C V M L K S R G S ~ G H V R F O D A F C G A Y F T F A l S H E G H V Y - G F G L S - N Y H P L G T - P G T E - S C F I - P Q N L T S F K N S T K S W V - - - - - - - - - - - - - - - - - - G F S G G P H H T V C M D S E G K A Y - S L G R A - E Y G R L G L G E G A E E K S - l - P - - - - T L l - S R L P A V S - - - - - - - - - - - - - - - - - S V A C G A S V G Y A V T K D G R V F FIGURE 6.-ATSI has structural - A W G M G T N Y - P L G T G Q D E D A U S - - - P V E M M G K Q - L E N R V V L - - - - - - - - - - - - - - - - - S V S S G G ~ H T V L L V K D K E Q S ~ and functional similarity to the

- G C G - D N R R G E L D S A Q A L R P V H D U R P - - - - - - V E V P A P V V - - - - - - - - - - - - - - - - - - D V A C G U D T T V I V D ~ D G R V U . * . I . . ...... ............... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .*. . f

* * . . t .

B YCpSO pDK7 (TUB])

RCCl family of proteins. (A) Com- parison of an A TSl protein repeat with the repeats in RCCl. The RCCl protein is depicted at the top, arranged to demonstrate its seven tandem repeats. The second ATSI repeat (residue 59 to residue 109) is lined up under the RCCl repeats. The number of stars under each ATSl amino acid indicates the number of times that particular Atslp residue occurs in an RCCl repeat. Comparison of amino acids that are highly conserved in RCCl to conserved ATSl residues (Figure 2) demon- strates that these amino acids are conserved in both proteins. (B) PRp20 is capable of suppressing the cold sensitivity associated with

graphs of plates containing transformants of DBY2412 (tubl-730) grown at the nonpermissive temperature of 15" for 2 weeks. The upper left shows DBY2412 transformed with YCp50, the upper right DBY2412 transformed with pDK7 ( CEN TUB1 ), the lower left DBY2412 transformed with pDK32 (2-pm ATSl ) and pWF48 (2ym PRp20).

DBY2412 (tubl-730). Photo-

pDK32 (2-pm ATSI) pWF48 (2-pm fRf.70)

the mutant being suppressed. A quantitative suppressor of mutants exhibiting excess microtubules might act by reducing the number or length of microtubules in the cell. Such a protein could suppress by decreasing microtubule stability, severing microtubule polymers, or altering the association constants between dimers and the ends of microtubules. Our data suggest that ATSl does not suppress the cold sensitivity of class 2 mutations by promoting microtubule disassembly: microtubules are not altered in wild-type cells that contain excess ATSl and defects of the class 1 no-microtubule mutants are not exacerbated by overexpression of A TSI. Finally, &tubulin strains that exhibit excess microtubules are unaffected by A T S l , indicating that ATSI does not act by reducing microtubule number or length.

An alternative mechanism is suggested by the exten- sive homology of A TSI with RCCl and related genes. All of these genes (human RCCI (OHTSUBO et al . 1987), S. cerevisiae SRMl/PRP20 (AEBI et al. 1990), Schizo-

saccharomyces pombe p iml (MATSUMOTO and BEACH 1991a,b), Drosophila BJI (FRASCH 1991) and Xenopus RCCl (NISHITANI et al. 1990)) encode proteins which can be arranged as seven tandem repeats, with each repeat consisting of 50-60 amino acids. A number of re- searchers have speculated that RCCl acts as a checkpoint protein (see for example, HARTWELL and WEINERT 1989; MURRAY 1992; NISHIMOTO et al. 1992), monitoring the state of DNA synthesis in the cell and preventing premature induction of mitosis. Cell lines mutant in RCCl prematurely condense their chromosomes upon shift to nonpermissive temperatures; a similar condensation phenotype is seen in the S. pombe gene, p iml (MATSUMOTO and BEACH 1991a,b). Mutants in SRMI/ PRP20 have been reported to have an increased level of chromosome missegregation (CLARK et al. 1991). RCCl homologs have been shown to interact by biochemical or genetic criteria with a ras-like protein: (1) RCCl can function as a guanine-nucleotide exchange factor for


TABLE 3

Percentage similarity between Atslp and Prp20p repeats

ATSl 1 ATSl 2 ATSl 3 ATSl 4 ATSl 5 ATSI 6

ATSl 7 53.7 44.7 48.6 51.2 36.8 52.8 ATSl 6 60.5 50.0 36.6 57.5 ATSI 5 26.5 47.4 44.4 41.7 ATSl 4 50.0 46.0 48.6 ATSI 3 40.5 48.7 ATSI 2 50.0

52.8

PRP20 1 PRP20 2 PRP20 3 PRPZO 4 PRP20 5 PRP20 6 PRP20 7

ATSl 7 39.5 47.4 33.3 40.5 44.2 39.0 40.5 ATSl 6 50.0 29.8 62.9 47.7 54.1 48.7 39.5 ATSl 5 42.5 40.5 47.5 42.4 37.5 50.0 48.4 ATSl 4 47.1 55.1 49.0 38.5 43.1 28.6 42.0 ATSl 3 60.0 41.5 44.4 40.0 43.9 48.6 44.4 ATSl 2 44.9 43.1 48.9 47.7 39.2 39.2 44.2 ATSl 1 50.0 57.7 51.9 47.2 61.5 52.0 55.8

PRP20 1 PRP20 2 PRP20 3 PRP20 4 PRP20 5 PW20 6

PRP20 7 49.0 54.0 39.0 45.4 54.0 53.2 PRP20 6 58.2 47.6 59.6 45.8 62.3 PRPZO 5 56.4 58.9 45.1 52.9 PRPZO 4 52.0 48.1 41.2 PRPZO 3 55.1 49.1 PRP20 2 54.4

Percentage similarity between Atslp and Prp20p repeats. Similarities were generated using the Genetics Computer Group program “Bestfit.” Settings for the program were the standard default settings. Boldface percentages indicate the highest and lowest percentage within that comparison group.

Ran/TC4, the mammalian ras-like protein (BISCHOFF and PONSTINGL 1991a,b) ; (2) spil, the S. pombe Ran/TC4 homolog, is a high copy suppressor of piml ( MATSUMOTO and BEACH 1991b) and (3) GSPl and GSP2, high copy suppressors of P920-1 in S. wmisiae, are Fbn/TC4 homologs (BELHUMEUR et al. 1993). Ran has recently been implicated in nuclear transport in Xenopus (MOORE and BLOBEL 1993). RCCl is capable of binding to chromatin (DMSO et al. 1992). S. cerevisiae is the first organism in which two Ran/TC4 homologs and two RCCI-like genes (ATSI and PRP20) have been identified.

A model of ATSl suppression that is consistent with the data generated in our study takes into account the data concerning RCCl and related proteins. The mutant a-tubulin produced by the class 2 tubl mutants could cause the cells to arrest at a point in the cell cycle near mitosis, leading to the large budded phenotype. Failure to form colonies at the nonpermissive temperature may be due to the cell cycle arrest. Suppression of this block might occur when overproduction of ATSl overrides the signal being generated by the mutant tubl protein, either directly or indirectly, allowing transit through the cell cycle to resume. By this model, the excess microtubule morphology observed would be a consequence of the extended arrest in the cell cycle.

One prediction of such a model would be that re- moval of ATSl in the class 2 tubl mutants would be deleterious, as it would take away the already present basal level of ATSl protein which is presumably mod- erating the strength of the signal generated by the mu-

tant Tublp. No strong synthetic phenotype is detected in strains mutant for both TUB1 and ATSl. However, alterations in growth rate are observed in strains doubly mutant for an ATSl deletion and tubl-714. The lack of a strong phenotype may be due to a functional overlap between ATSl and another gene, perhaps PRPZO.

The model explains why A TSl overexpression has no detectable phenotype: cells normally do not arrest at the Atslp execution point and so do not produce a substrate upon which the protein can act. Class 1 no-microtubule mutant cells arrest with a large budded phenotype reminiscent of the class 2 mutants, but overexpression of ATSl in these class 1 mutants has no effect. From this result, it appears that class 1 and class 2 tubl mutants, although blocked at similar stages in the cell cycle as assessed by bud morphology, are not arrested at the same point, rendering excess ATSl ineffective. Perhaps the reason that the Ptubulin excess microtubule mutants are not suppressed by overproduction of ATSl is that they also are not blocked at an appropriate stage.

Many aspects of this model are exhibited by a class of proteins referred to as checkpoint proteins (HARTWELL and WEINERT 1989). These proteins act to regulate tran- sitions through the cell cycle, responding to signals generated by factors or events that interact with specific stages of the cycle. Coordination of cell cycle timing is achieved by monitoring early events to ensure that they are completed before initiation of later events depend- ent upon those early steps. Checkpoint proteins need not be essential; the feedback control may not be

High Copy Tubulin Suppression 39 1

required in every cell cycle transit, due to the tendency of the cell cycle to continue unless perturbed (as dis- cussed in MURRAY 1992).

Although PRP20 overexpression is capable of s u p pressing the cold-sensitivity associated with the class 2 tubl mutants in a manner similar to ATSl, functionally the two genes are not completely interchangeable. Over- expression of ATSl in prp20-2 temperature-sensitive strains at a nonpermissive temperature does not rescue the cells, while deletion of ATSl does not alter the phenotypes of the prp20-2 mutant. Mutations in PRP20 have pleiotropic phenotypes. They have been isolated as defective in RNA polyA-tail processing and splicing (AEBI

et al. 1990; FLEISCHMANN et aZ. 1991), in addition to acting to allow mating in a MATa strain lacking the STE3receptor (CLARK and SPRAGUE 1989). However, these phenotypes need not be a direct consequence of loss-of-function of this essential gene, but rather secondary consequences.

It is intriguing that suppression of mutant tubl alleles by overexpression of wild-type genes has, in two separate cases, pointed to links between the microtubule and regulation of the cell cycle. In addition to this report, suppression of class 1 no-microtubule tubl mutants has led to the isolation of BUB3, a previously identified gene thought to encode a checkpoint protein in S. cereuisiae (Hour et al. 1991). Overexpression of BUB3 in a subset of the class 1 mutants, those that have an alanine to valine substitution at position 422, rescues the cold sensitivity associated with the mutation (S. GUENETTE, M. MACENDANTZ and F. SOLOMON, submitted for publication). As the state of the cell cycle and the control of the action of microtubules must by necessity be tightly coordinated, more interactions of this nature may very well be uncovered.

We thank W. FORRESTER and M. WICKENS (University of Wisconsin at Madison) for the kind gift of pWF48 (2ym PRP20), theprp20-2 strain and conversations on unpublished data concerning PRPPO. We also thank T. HUFFAKER and G. BARNES for the tub2 strains used in this study. Finally, we thank the past and present members of the SOLOMON laboratory for innumerable discussions and advice during the course of this work. This work was supported by National Institutes of Health Grant 5-R01-GM41477-04 and by National Institutes of Health Training Grant 5.T32-GM07287 to the Massachusetts Institute of Technology.

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Communicating editor: S. JINKS-ROBERTSON

yeast homologs of the mammalian checkpoint …pressing element, called a tsl (for alpha tubulin...

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